Expanding structural diversity of 5′-aminouridine moiety of sansanmycin via mutational biosynthesis

Sansanmycins represent a family of uridyl peptide antibiotics with antimicrobial activity specifically against Mycobacterium tuberculosis (including drug-resistant M. tuberculosis) and Pseudomonas aeruginosa. They target translocase I (MraY) to inhibit bacterial cell wall assembly. Given the unique mechanism of action, sansanmycin has emerged as a potential lead compound for developing new anti-tuberculosis drugs, while the 5′-aminouridine moiety plays a crucial role in the pharmacophore of sansanmycin. For expanding the structural diversity of the 5′-aminouridine moiety of sansanmycin through biosynthetic methods, we firstly demonstrated that SsaM and SsaK are responsible for the biosynthesis of the 5′-aminouridine moiety of sansanmycin in vivo. Using the ssaK deletion mutant (SS/KKO), we efficiently obtained a series of new analogues with modified 5′-aminouridine moieties through mutational biosynthesis. Based on molecular networking analysis of MS/MS, twenty-two new analogues (SS-KK-1 to -13 and SS-KK-A to -I) were identified. Among them, four new analogues (SS-KK-1 to -3 and SS-KK-C) were purified and bioassayed. SS-KK-2 showed better antibacterial activity against E. coli ΔtolC than the parent compound sansanmycin A. SS-KK-3 showed the same anti-TB activity as sansanmycin A against M. tuberculosis H37Rv as well as clinically isolated, drug-sensitive and multidrug-resistant M. tuberculosis strains. Furthermore, SS-KK-3 exhibited significantly improved structural stability compared to sansanmycin A. The results suggested that mutasynthesis is an effective and practical strategy for expanding the structural diversity of 5′-aminouridine moiety in sansanmycin.

SS-KK-3 have the same AA1 and AA3 with SS-KK-1/2 based on their consistent characteristic fragment ions of F5 and F9, but with a total mass weight 23 Da lower than that of SS-KK-1, implying that the AA4 of SS-KK-3 is (m-)Tyr, which has a molecular weight of 23 Da lower than that of tryptophan (Trp).SS-KK-4, SS-KK-5 and SS-KK-8 were found that only AA1 was altered compared to SS-KK-1/2 based on the same characteristic fragment ions of F2, F5, F7, and F8.The AA1 of SS-KK-4 was 12 Da larger than that of m-Tyr, indicating the presence of a bicyclic a/b which was previously found in sansanmycin K and J (Xie et al., 2014).In SS-KK-5, AA1 was 42 Da larger than m-Tyr, possibly due to an additional acetylation, resulting in N-Acetyl-(m-)Tyr.The AA1 of SS-KK-8 was 57 Da larger than that of m-Tyr, which is speculated to be an additional glycine (Gly) substitution resulting N-Gly-(m-)Tyr.
SS-KK-6 has a molecular weight of 16 Da larger than SS-KK-1, suggesting that they may have one more oxygen atom.The AA3 of SS-KK-1 might be replaced by oxidized methionine (Met SO ) rather than methionine (Met).This hypothesis was also supported by the characteristic losses of methylsulfonyl group (64 Da) of m/z 818 of M−64 and m/z 614 of F10−64.

SS-KK-7 exhibited molecular ion [M+H]
+ at m/z 894, 16 Da more than that of SS-KK-4.All the characteristic fragments related to AA3 (F2, F5, and F10) are also 16 Da more than those in SS-KK-4, suggesting that it has an additional oxygen atom compared to SS-KK-4 at the AA3 position.
The quasi-molecular ion [M+H] + of SS-KK-9 at m/z 839 was 39 Da lower than that of SS-KK-4.The tandem mass spectrometry analysis confirmed the presence of a consistent characteristic fragment ion at m/z 674 (F10), indicating that the AA4 was changed from Trp in SS-KK-4 to Phe in SS-KK-9.
The quasi-molecular of SS-KK-10 was 18 Da lower than that of SS-KK-1.After in-depth analysis, it was found that the change occurs in AA3 due to the F7 and F10 of SS-KK-10 both lower than those of SS-KK-1.Therefore, AA3 was speculated to be leucine (Leu) with a molecular weight 18 Da lower than that of Met.SS-KK-11 has a molecular weight of 854, 12 Da larger than that of SS-KK-3.While both compounds showed consistent F7 ion at m/z 339, the F3 ion at m/z 207 in SS-KK-11 was 12 Da greater than the F3 ion at m/z 195 observed in SS-KK-3.Thus, it can be inferred that AA1 of SS-KK-11 was bicyclic a/b.
The tandem mass spectrum of SS-KK-12 differs significantly from the resolved sansanmycins.It was speculated that AA3 of SS-KK-12 is Met SO because of the appearance of diagnostic fragments ion of m/z 835 (M-64).Characteristic fragment of F1 ion at m/z 176, corresponding to the residue of AA1, was 28 Da larger than that of bicyclic a/b, suggesting that the AA1 of SS-KK-12 is bicyclic d with two more methyl groups compared to bicyclic a/b.Based on this, AA4 was presumed to be m-Tyr/Tyr ((m-)Tyr) according to the quasi-molecular ion of SS-KK-12.The remaining characteristic fragment ions also supports this inference.
The quasi-molecular of SS-KK-13 was 16 Da larger than that of SS-KK-5.Combining with diagnostic fragment ion at m/z 655 (F2-64), the AA3 SS-KK-13 was inferred to be Met SO .
1.1.2New sansanmycin derivatives generated by feeding precursor 2 The molecular weight of 2 was 14 Da larger than that of 1.Similarly, the quasi molecular of SS-KK-C, SS-KK-F and SS-KK-H were 14 Da larger than that of SS-KK-4, SS-KK-6 and SS-KK-12, respectively.It is speculated that SS-KK-C, SS-KK-F and SS-KK-H share the same amino acids as SS-KK-4, SS-KK-6 and SS-KK-12, respectively, only uridine was replaced by 2. This inference was also supported by the corresponding tandem mass spectrum fragments.SS-KK-D has a molecular weight of 16 Da larger than SS-KK-C, suggesting the AA3 of SS-KK-D may be Met so , which is 16 Da larger than Met.This hypothesis was also confirmed by characteristic fragments of m/z 844 (M-64) and m/z 640 (F10-64).
The molecular weight of SS-KK-E was 28 Da higher than that of SS-KK-C, likely due to two additional methyl substitutions on AA1, which suggests AA1 is bicyclic d.The consistent fragments of F2 ions at m/z 717 indicates the change only occurs on AA1, while the characteristic fragment of F1 ion at m/z 176, corresponding to the residue of bicyclic d, further supports this inference.
The quasi-molecular of SS-KK-G was 14 Da larger than that of SS-KK-C, possibly because of an extra methyl substitution.Both compounds share the same AA3 and AA4 as evidenced by the identical fragments of F7 ion at m/z 362.This suggests that the AA1 in SS-KK-G may be bicyclic c, which has one more methyl substitution than bicyclic a/b, and was previously found in sansanmycins (Jiang et al., 2018).
The quasi-molecular of SS-KK-I showed 18 Da lower than that of SS-KK-E, and it can be inferred that they contain the same AA1 due to the same ions of m/z 176 (F1) and m/z 235 (F3).F10 ion of m/z 698 in SS-KK-I was 18 Da lower than that of SS-KK-E, indicating that the AA3 of SS-KK-I should be Leu rather than Met.The spectra were recorded in DMSO-d6.The chemical shifts (δ) are given in ppm.

Figure S3 .
Figure S3.Sequence alignment of SsaK and PacK.

Figure S8 .
Figure S8.The structures of uridine analogues fed to SS/KKO.

Figure S9 .
Figure S9.Molecular network consisting of all parent ions detected by LC-MS in the extract crude of SS/KKO when fed with 1 or 2.